JP2009531696A - Improved SERRS base material - Google Patents

Abstract

Depositing a Raman enhancing surface on or within a porous three-dimensional support matrix of solid support material provides an improved SERRS substrate for use in an improved analyte detector. The support material is configured to have a Raman dye dispersed within the volume, as a result of the dye being very close to the Raman enhancing surface that is dispersed within the volume and similarly dispersed within the volume. , The response to dye irradiation is enhanced. A reaction carrier is provided into which a test sample can be introduced for use in a detector configuration that detects the presence, absence, or amount of an analyte in the sample.

Description

  The present invention relates to Raman spectroscopy, surface enhanced Raman spectroscopy, and surface enhanced resonance Raman spectroscopy.

It is well known that there are many techniques for detecting the action or presence of an analyte molecule. One such technique utilizes the Raman Scattering (RS) effect. When light is scattered from the molecule, most of the photons are elastically scattered. Most of the scattered photons have the same energy (and therefore frequency and wavelength) as the incident photons. However, a small amount of light (about 1 in 10 ⁷ photons) is scattered at a different frequency than the incident photons. When the scattered photon loses energy to the molecule, it has a longer wavelength than the incident photon (referred to as Stokes scattering). Conversely, when a scattered photon gains energy, it has a shorter wavelength (referred to as anti-Stokes scattering). Stokes scattering usually has a stronger effect.

  The process leading to this inelastic scattering was first described in 1928 by Sir C.C. V. From Raman, it is called the Raman effect. This is related to molecular vibrations, rotations, or changes in electronic energy, and the energy transferred from the photon to the molecule is usually dissipated as heat. The energy difference between the incident photon and the Raman scattered photon is equal to the energy of the vibrational state of the scattered molecule or of the electronic transition, resulting in a scattered photon in the quantization energy difference with the incident laser. A plot of scattered light intensity versus energy or wavelength difference is referred to as a Raman spectrum and the technique is known as Raman Spectroscopy (RS).

  Surface-enhanced Raman spectroscopy (SERS) is a variation of the RS analysis technique. When a molecule is in physical proximity to a particular metal surface, further energy transfer between the molecule and the surface electrons (plasmons) of the metal can greatly increase the intensity of the Raman signal. In order to perform SERS, analyte molecules are absorbed on a substrate with an atomically roughened metal surface and enhanced Raman scattering is detected. Moreover, SERS can be enforced even if it uses the silver colloid in a solution as a base material.

Raman scattering from molecules or ions in metal surfaces of several angstroms can be 10 ³ to 10 ⁶ times greater in solution. For near visible wavelengths, SERS is strongest on silver but can also be easily observed on gold and copper. Recent studies have shown that a variety of other transition metals can also provide useful SERS enhancement. So-called free electron metals, which are metals with large amounts of free surface electrons, generally provide SERS enhancement. Furthermore, so-called metal polymers can also be used. These are organic polymers with electronic structures that behave like metals. It will be understood that the term metal is not limited to metal elements or metal mixtures or alloys, but is applicable to any material that can be understood by a person skilled in the art to be a metal. Materials that provide such SERS enhancement are hereinafter referred to as Raman-enhanced surfaces or Raman-enhanced metals.

  In essence, the SERS effect is a resonant energy transfer between a molecule and an electromagnetic field near the metal surface. The electric vector of the excitation laser induces a dipole in the metal surface, and its restoring force becomes an oscillating electromagnetic field at the resonance frequency of this excitation. This resonance intensity and frequency is mainly determined by the free electrons (“plasmons”) on the metal surface that determine the so-called plasmon wavelength and the dielectric constant of the metal and its environment. Molecules that are absorbed on or very close to the surface experience a very large electromagnetic field, which is most significantly enhanced in coupling to vibrational modes that are perpendicular to the surface. This is a surface plasmon resonance (SPR) effect that allows energy transfer through the space between plasmons and molecules near the surface. Since energy transfer efficiency depends on a good match between the laser wavelength and the plasma wavelength of the metal, the intensity of the surface plasmon resonance depends on many factors, including the wavelength of the incident light and the morphology of the metal surface.

  The strength and local density of the electromagnetic field is determined by various parameters. The wavelength of the reflected light determines its energy, and the composition and morphology of the metal determines the intensity and efficiency with which surface plasmons combine with the photon energy. Other factors such as the relative dielectric properties of the metal and analyte solution also contribute significantly to the effect. In addition, the energy transfer efficiency between the electromagnetic field near the metal surface and any molecule is also determined by the resonance energy state of the molecule itself, including, for example, specific vibrational modes in the infrared spectral region and the electronic energy transition of ultraviolet light. Is done. This is the mechanism by which SERRS obtains better performance than conventional SERS. SERRS can be performed using a chromophore moiety and provide an additional molecular resonance contribution to energy transfer.

The intensity of the resonance Raman peak is proportional to the square of the scattering cross section of the molecule. And the scattering cross section is related to the square of the transition dipole moment and therefore usually follows the absorption spectrum. If the incident photon has an energy close to the absorption peak in its absorbance spectrum, a scattering event can cause the molecule to become more excited, thereby increasing the relative strength of the anti-Stokes signal. The combination of surface and resonance enhancement effect means that SERRS can provide enormous signal enhancement, typically 10 ⁹ to 10 ¹⁴ times that of conventional Raman spectroscopy.

  In addition to resonance enhancement of Raman scattering, resonance attenuation has recently been reported that reduces the intensity of the Raman signal by a resonant energy transfer mechanism. Under specific conditions, an excitation energy state whose energy is close to the target excitation energy state may cause a reduction in Raman scattering. In this case, the Raman intensity is proportional to the square of the sum of the cross-sectional areas, and if they are of opposite sign, destructive interference occurs, resulting in the observed resonance attenuation. This provides an alternative metric for use in Raman-based detector systems. Signals from specific Raman-active chromophores can be selectively removed from the Raman spectrum using laser frequencies that promote this damping effect.

  Surface-enhanced Raman spectroscopy (SERS) and its extension, surface-enhanced resonance Raman spectroscopy (SERRS), are attracting attention as quantitative bioanalytical tools. Both techniques rely heavily on the interaction between the “plasma” of mobile conduction electrons (plasmons) on the metal surface and the molecular species near the surface. This interaction results in a large enhancement of Raman scattering at specific vibrational energy, producing a strong spectral signal in the Raman scattered light.

Until recently, discussions had been held over the understanding of the augmentation mechanism. The distribution of the enhancement factor of greater than 10 ⁶ between the chemical enhancement mechanism and the electromagnetic enhancement mechanism, the two views of the major factions were divided. Chemopotentiating mechanism currently believed to contribute to the enhancement factor of 10 ^2, a charge transfer state claims be generated between the adsorbate molecules and metal. This mechanism is site specific and analyte dependent. Molecules must be absorbed directly onto the surface to experience chemical enhancement. Electromagnetic enhancement mechanism contributes to enhancement of more than 10 ⁴ times greater than normal Raman scattering. In order to understand electromagnetic enhancement, the size, shape, and material of the surface nanoscale roughness properties must be considered. If the correct wavelength of light strikes the metal roughness characteristics, the conduction electron plasma will oscillate collectively. Since this collective vibration is localized on the plasma surface of electrons, it is known as Localized Surface Plasmon Resonance (LSPR). LSPR absorbs and scatters resonant wavelengths and creates a large electromagnetic field around the roughness characteristics. When a molecule is placed in an electromagnetic field, an enhanced Raman signal is measured.

  The inventors have found that the Raman scattering effect provides only a small amount of Raman scattered radiation compared to normal scattering (effectively, using Surface Enhanced Raman Scattering; SERS). Understand that the small signal-to-noise ratio is a problem. Further, it will be appreciated that because the Raman signal is weak compared to noise, there is a need to introduce a mechanism that helps distinguish the Raman signal from noise.

  It will be appreciated that reducing the diffusion path length of analyte molecules can have a dramatic effect on the speed of the biosensor assay. Thus, in a broad sense, the present invention provides an arrangement that reduces the diffusion path length of a sample that is tested using spectroscopic techniques.

  The invention is defined in the claims for reference.

  Embodiments of the present invention provide improved analyte detection having a Raman enhanced volume located within a reaction region generated by depositing a Raman enhanced surface on or within a porous three-dimensional support matrix of solid support material Provide a bowl. The support material is configured to have a dye dispersed within the volume, the dye being dispersed within the volume, and as a result of being very close to the Raman-enhanced surface that is similarly dispersed within the volume, against dye irradiation. The response is enhanced.

  A reaction carrier for use in an analyte detector into which a test sample is introduced is produced in accordance with the present invention having a solid support material defining a volume and a metal / Raman enhanced surface dispersed and immobilized within the volume. May be. The metal is supported by a support material that is porous to the dye that makes the analyte detectable. The metal is configured to enhance the light response to dye irradiation.

  Although the described embodiments of the present invention refer to improvements provided for Raman signals and their application in Raman spectroscopy (particularly SERS and SERRS), the present invention may be used in any form of spectroscopy. It should be understood that improvements in signal intensity may be obtained using metal surfaces that enhance the response of dye irradiation. This may include, for example, surface-absorbed fluorescence or any response to irradiation with resonance energy transfer to the metal surface.

  The term chromophore is well known to those skilled in the art and is used herein to cover groups having specific optical properties. The term “dye” refers to a chromophore that is capable of emitting Raman radiation and that has some functionality such as a linking group or surface exploration group. Such functionality can arise, for example, from the addition of metal surface probing groups or groups that allow binding to the analyte. The chromophore should absorb the excitation laser at a wavelength suitable for surface enhancement (most used Raman lasers are 514 nm, 532 nm, and 785 nm). Since this is in the red-green visible region, the conventional bright colored chromophore is recognized as a particularly good component of the Raman active dye.

  An analyte is any chemical substance that is desired to be detected or quantified. Examples of suitable analytes include biomolecules (proteins, antibodies, nucleic acids, carbohydrates, proteoglycans, lipids, or hormones), drugs or other therapeutic agents and their metabolites, drugs of abuse (eg, amphetamines, opiates, benzodiazepines) , Barbiturates, cannabinoids, cocaine, LSD, and metabolites thereof), explosives (eg, nitroglycerin and nitrotoluene, including TNT, RDX, PETN, and HMX), and environmental pollutants (eg, herbicides, insecticides) Agent).

  An analyte sample is any sample that is desirable for testing the presence or amount of an analyte. There are many situations that are desirable for testing for the presence, absence, or quantity of an analyte. Examples include clinical applications (eg, to detect the presence of antigens or antibodies in biomolecular samples such as blood or urine samples), detection of the presence of drugs of abuse (eg, illegal samples, or fluid or breath samples) Detection of explosives, or detection of environmental pollutants (eg, in liquid, gas, soil, or plant samples).

  In addition to directly detecting the analyte itself, it is also possible to detect them indirectly by using a reporter molecule that can generate a detectable change in its Raman signal in the presence of the analyte of interest. It is. An example of this is the displacement of the dye-labeled peptide from the antigen-binding site of the analyte-specific antibody, whereby the free reporter molecule is released and can then interact with the SERRS active metal surface. For our purposes, such reporter molecules can also be regarded as “analytes”.

  Typically, the reporter molecule will comprise a reporter dye, a selective agent binding group, and a metal surface binding group. The reporter molecule is bound to the selective agent (via the selective agent binding group), so that the reporter dye is held away from the metal surface before the analyte sample is introduced to the carrier. Binding of the analyte to the selection agent displaces the reporter molecule, which then binds to the metal surface (via the metal surface binding group), thereby moving the reporter dye to a region near the metal surface. The term “dye” described above applies equally to reporter molecules.

  A selective agent is any agent that selectively binds to an analyte in the presence of other components of the analyte sample, and further under conditions such that the detection method is performed to detect the presence (or amount) of the analyte in the sample. It is an agent. Of course, the nature of the selective agent will depend on the identity of the analyte. In many cases, the selective agent will be an antibody. However, other suitable analyte binding partners may be used. For example, when the analyte is an antibody, the selection agent may be an antigen or an antigen derivative that is selectively bound by an antibody. When the sample is a nucleic acid, the selection agent may be a nucleic acid or a nucleic acid analog that is hybridized to the sample nucleic acid.

  The dye need not be removed from the selective agent upon introduction of the analyte. Alternatively, the selective agent may change configuration in response to analyte binding so that the dye moves to a new location closer to the metal, thereby resulting in an increase in the Raman signal. It is also possible to initially hold the dye close enough on the metal surface to generate a SERRS signal, but it may be displaced in the presence of the analyte to be detected, eg The dye may be displaced from its position near the metal surface. In this case, rather than an increase in the emitted Raman radiation, a decrease in the Raman radiation is expected that makes it possible to determine its absence, presence or amount.

  The specimen itself may be essentially Raman active. In such embodiments, the dye may be chemically equivalent to the analyte, and the presence, absence, or amount of the analyte can be determined directly from its Raman signal. Thus, the term “dye” may include an analyte.

  The term “antibody” selectively binds an antibody or fragment (eg, Fab fragment, Fd fragment, Fv fragment, dAb fragment, F (ab ′) 2 fragment, single chain Fv molecule, or CDR region), or analyte. As used herein to include derivatives of antibodies or fragments that allow detection of analytes.

  In general, the components of the dye are expected to be bound together by separate linkers. One skilled in the art will appreciate that there are many possible suitable linkers that can be used. The identity of the linker will depend on the identity of the dye components. Where the selective agent binding group comprises a peptide, it is advantageous if the linker is compatible with conventional peptide bond chemistry. For example, the linker may preferably be equipped with a single carboxylic acid group for reaction with the N-terminus of the peptide.

  In some situations, depending on the particular component used, two or more components of the dye may be joined together, for example, by reaction between chemical groups of different components of the dye, without using a separate linker. It may be possible.

  The dye components may be bonded together in any order, provided that the dye is in a region near the metal surface if the dye is bonded to the surface by its metal surface binding group.

  The metal surface binding group of the dye should be a group that binds preferentially to the metal surface (typically by absorption). In certain situations, it may be desirable to bond the metal surface binding group to the metal surface to be strong enough to fix the dye to the metal surface. The chemical nature of the metal surface binding group will depend on the metal surface used. Suitable silver binding functional groups are oxazole, thiazole, diazole, triazole, oxadiazole, thiadiazole, oxathiazole, thiatriazole, benzotriazole, tetrazole, benzimidazole, indazole, isoindazole, benzodiazole, or benzisodiazole A group having a heterocyclic nitrogen, and the like. Other suitable functional groups include amines, amides, thiols, sulfates, thiosulfates, phosphates, thiophosphates, hydroxyl groups, carbonyls, carboxylates, and thiocarbamates. Amino acids such as cysteine, histidine, lysine, arginine, aspartic acid, glutamic acid, glutamine, or arginine also impart silver bonds.

  The term reaction carrier is used to define the container into which the analyte to be detected is introduced and where the support material is located.

  DETAILED DESCRIPTION Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings.

  The described embodiment features an improved substrate for use in spectroscopy. The present invention uses a metal dispersed within a particulate three-dimensional support material to produce a substrate with SERS and SERRS optimal properties. By making the metal physically closer to the analyte molecules in the solution, the diffusion path length can be reduced, the assay time can be shortened, and the analyte molecules in the given solution volume can be accessed Increase possible metal surface area. In addition, the Raman irradiation laser samples a three-dimensional volume, not a two-dimensional surface, and has an effect of solving not only signal intensity but also engineering problems such as focusing. Also, the engineering constraints in supporting the biosensor chip can be somewhat relaxed because it is the nature of the support material rather than the dimensions of the reaction support that most affect sensor performance.

  Embodiments of the present invention are realized by chemically depositing a portion of the metal in the support material. Some effects that can be achieved by this include enhanced sensitivity, long-term stability of the photolabile chromophore, and greatly reduced assay time. As a preferred property, the metal portion deposited in the support material that forms the Raman enhancing surface is silver. The support material is, for example, a solid in the form of a silica filter, but this is not a requirement and the support material may be, for example, in the form of powder or glass beads. The main requirement of the support material is to maintain the fixation of the Raman enhancing surface within the reaction support.

  Next, an improved substrate was developed using silver deposition chemistry based on an analytical test for carbohydrates, developed by German chemist Bernhard Tollens (1841-1918) and now named him. A generation method will be described. Those skilled in the art will appreciate that this is not the only means of depositing metal on a surface, and that other methods are within the scope of the present invention. In addition, although silver exhibits many desirable properties for use as a Raman enhancing surface in spectroscopy, it will be understood that other materials such as gold and copper can also be used. Also, the use of combinations of these metals can imply advantages in the flexibility of the present invention, which are described below.

  The Tollens reaction is a multi-step process that involves the oxidation of aldehydes to carboxylic acids and the reduction of mercury-containing ions to metals. First, silver hydroxide is prepared from silver nitrate by reacting with sodium hydroxide.

水酸化物は、通常、酸化銀も含有する黒褐色沈殿物を形成する。水酸化アンモニウムを添加し、無色溶液として、ジアミン銀錯体を生成する。

The hydroxide usually forms a dark brown precipitate that also contains silver oxide. Ammonium hydroxide is added to form a diamine silver complex as a colorless solution.

ジアミン錯体は、水溶液中で安定するため、必要となるまで保存可能である。銀堆積プロセスは、アルデヒドを添加することによって開始する。アルデヒドは、アルカリ溶液中で水酸基イオンと反応し、カルボン酸に酸化させ、２つの電子を放出する。

Since the diamine complex is stable in an aqueous solution, it can be stored until needed. The silver deposition process begins by adding an aldehyde. Aldehydes react with hydroxyl ions in an alkaline solution, oxidize to carboxylic acid and release two electrons.

アルデヒドは、種々の源から得られる。従来のＴｏｌｌｅｎｓ反応では、グルコース溶液が使用される。多くの糖類同様、グルコースは、閉環または開鎖形態で存在可能である。水溶液中では、両形態は、平衡である。

Aldehydes are obtained from a variety of sources. In the conventional Tollens reaction, a glucose solution is used. Like many sugars, glucose can exist in a closed or open chain form. In aqueous solution, both forms are in equilibrium.

次いで、アルデヒドの酸化によって放出された電子は、それぞれジアミン銀錯体を還元し、金属銀および遊離アンモニアを提供し得る。

The electrons released by oxidation of the aldehyde can then reduce the diamine silver complex, respectively, to provide metallic silver and free ammonia.

この酸化還元反応は、以下のように集約され得る。

This redox reaction can be summarized as follows.

還元銀原子は、水溶液中で不安定であるため、迅速に凝集し、金属銀を提供する。適切な固体表面が利用可能な場合、金属が表面と溶液との間の界面で形成される。糖類の従来のＴｏｌｌｅｎｓ試験では、清潔なガラス表面が使用され、銀によって融合性の鏡状フィルムがわずか数分間で形成される。

Since the reduced silver atoms are unstable in aqueous solution, they rapidly aggregate and provide metallic silver. If a suitable solid surface is available, metal is formed at the interface between the surface and the solution. In the traditional Tollens test for sugars, a clean glass surface is used and a fusible mirror film is formed by silver in just a few minutes.

  Depending on the specific reaction conditions that depend on the analyte of interest and the nature of the support material available, Raman-enhanced surfaces (preferably silver) are present on micro to nanometer-scale particles, particle populations, support materials and within Can form a suspension of particles deposited in any pores that do.

  The process described above is only one way of producing the support material according to the invention. Other methods can also be employed including, for example, fixation of silver colloid on or in the support structure.

  The support material is preferably a particulate, porous, three-dimensional support matrix selected to have the optical properties of glass. For example, the support material may be in the form of glass beads that fill a portion of the reaction support. In particular, the support material should not be fluorescent (can provide unacceptable electromagnetic field interference to the Raman spectrum), ideally it is very biocompatible and shields the Raman signal from the dye As such, it should not contribute to a substantial Raman signal that is unique or at least not within the frequency range of the Raman response of the dye. For these purposes, silica is ideal, but it should be understood that other materials having similar properties may be used without departing from the scope of the present invention. Examples of other materials that can be used to form the support material include ceramic, plastic, or aerogel.

  The support material defines a volume in which the silver particles can be dispersed. In one embodiment, the silver particles are deposited on the outside of the individual substrate particles that comprise the support material. This example can be seen in FIGS. 11 and 12, wherein the silica-based particles that define the volume have silver particles deposited thereon. This means that silver particles are present in the pores (111, 121) between the particles comprising the support material. A further example is a support material consisting of a collection of glass balls having silver deposited thereon. The voids or pores between adjacent balls, or the substrate particles are porous and become a support material having silver particles fixed to the entire support material.

  Additional embodiments are described below in which the substrate particles themselves with the support material are porous to the dye and have silver particles deposited within their internal structure.

  Three-dimensional porous materials such as silica provide a volume with a wider internal surface area and thus a wider surface available for molecular absorption compared to a flat surface. Also, instead of irradiating a point on a planar surface, the incident laser further increases the surface area that can be responded by the Raman laser by irradiating a volume in the material. Both of these factors result in a significant increase in the number of SERRS active molecules in the irradiated light with the improvements that occur as a result of signal intensity.

  FIG. 8 shows a microfluidic SER® detector configuration embodying the present invention. The detector configuration consists of a reaction carrier containing a dye, an irradiation source in the form of a laser, and a detector. A three-dimensional porous material such as silica with metal particles deposited therein is provided in the reaction support. Since the laser responds to the volume, not the plane, accurate focusing on the sample becomes less important. Instead, as shown in FIG. 9, the laser beam is focused into the cone, passes through the focal point, and then expands into the cone beyond this point, so that the molecules immediately before and after the focal point are relatively It is in a strong electromagnetic field. In the ideal case, the number of molecules in the strong electromagnetic field should be doubled and therefore the Raman signal should be doubled when the focus is in the matrix as compared to when the focus is on a planar surface.

  The planar metal surface may act as a mirror and the pre-focus molecules will be in the enhanced EM electromagnetic field (from both incident light and its reflection). If the light beam interferes constructively, the molecule will be in an electromagnetic field that is twice as strong. Since the Raman intensity is proportional to the square of the electromagnetic field intensity, molecules in the double electromagnetic field emit four times the Raman signal. However, half of the molecules will not contribute to the Raman signal because they are in the region where the electromagnetic field interferes destructively. Therefore, the Raman scattering intensity as a whole will be twice that without the reflective surface. However, this signal will come from molecules in the electromagnetic field twice as a result, and as a result, will be photodegraded at a high rate. Photolysis occurs when the chromophore is permanently damaged due to photon-induced excitation and subsequent covalent modification. In response to a transition from an excited singlet state to an excited triplet state, the chromophore is likely to interact with another molecule to produce an irreversible covalent modification. The triplet state is relatively long-lived relative to the singlet state, thereby significantly increasing the chemical reaction time with components in the environment of the excited molecule. Thus, for a given surface area, the in-matrix focusing configuration can provide a Raman signal with the same intensity as the reflective surface, but gives the effect of reduced photolysis rate of the chromophore. If the surface is non-reflective, the matrix configuration will provide an enhancement signal.

  The particulate three-dimensional support matrix can be in the form of a silica filter frit (shown in the cross section of FIG. 10) and has a porous structure with tens of micrometers of pores between circular silica grains. . When used as a support surface for chemically deposited silver, the silica particles are coated with particulate metallic silver, as shown in FIG. 11 and schematically in FIG.

  The matrix pores 111/121 typically have a total length of about 50-80 μm and may have a diffusion path of 25-40 μm. For a typical 100 kDa protein analyte this would be 99% binding within about 30 seconds. The three-dimensional matrix provides the reaction rate advantage of microfluidic systems and at the same time allows detection volumes on the millimeter and higher scales due to relaxed requirements on laser focusing.

  The deposited metal part has two forms. The first form comprises the filamentary strand shown in FIG. 13, which is a fused linear chain of substantially spherical particles having a diameter of about 250 nm. These filaments are surrounded by a second form of smaller free particles in the tens to hundreds of nanometer diameter range, as shown in FIG. It should be understood that other forms formed from different methods may also work.

  FIG. 14 shows 510 generally spherical silver particles having a diameter in the range of about 12 nm to about 220 nm. The method required to produce this deposit involves completely immersing the filter frit in the Tollens reaction mixture.

  If the frit can float on the surface of the reaction mixture, the silver particles are deposited with a very high density per unit area, as shown in FIG. FIG. 15 presents a high particle surface density (976 in FIG. 15 compared to 510 in FIG. 14) but is produced by complete precipitation of the filter frit, as can be seen from the superimposed particle size distribution (FIG. 16). Have a particle size distribution very similar to that of The partial settling of the filter frit produces about twice as many particles per unit surface area as in Method 1 and thus provides a high Raman signal.

An exemplary SERRS spectrum of particles prepared on a silica filter and on a flat surface using two methods is shown in FIG. In all cases, the same SERRS active compound was used at a concentration of 10 ⁻⁵ M and an integration time of 1 second. Filtration method 1 shows the case where the frit has completely precipitated in the reaction mixture. Method 2 shows the case where the frit can float on the surface.

  The number of silver particles on the surface prepared using the floating frit is about twice, while the SERRS signal is about three times that of Method 1. This is because the signal intensity is not linearly dependent on the number of particles on the surface. Since the particles have the same particle size distribution, the non-proportionally increasing SERRS signal cannot simply be attributed to an increase in available silver surface and has an extra contributing factor due to the interaction between the particles. And higher packing density leads to further SERRS enhancement. It is generally accepted that particles interact via an electromagnetic field generated by the synchronized motion of conduction electrons therein, and this interaction depends on the distance between the particles.

  In further embodiments of the present invention, it is possible to further reduce the average diffusion path length of analyte molecules by depositing silver particles within the structure of the substrate particles comprising the support material itself. This can be achieved when the support material is porous. The term porous in this case refers to a substance that contains pores of a diameter such that the particles that make up the substance are porous to the dye used to detect the analyte. In this embodiment, the particles of the support material itself have pores or holes, resulting in a fully accessible internal structure.

  A good example of a support material composed of microporous particles is pore glass (CPG), which can be formed from silica. Microporosity indicates the fact that the pores have a diameter of about a few microns or tens of microns. One method of producing CPG is by acid etching of a glass mixture where one of the glasses is subject to acid corrosion but not the other. The product is a glass / silica material characterized by a fully accessible internal structure, where individual silica particles serve to increase the available internal surface area of silver particles adhering thereto Has pores of scale. Examples of such materials are shown in FIGS. 18-21.

  FIG. 18 shows an enlarged view of CPG in which individual particles can be confirmed. FIG. 19 shows the surface of a single particle, characterized by a number of interconnected pores and a surface providing a fully accessible internal structure. FIG. 20 shows a higher magnification view of the CPG particle surface with pore size (in this case several hundred nm), and FIG. 21 shows metal particles that can be deposited in the pores. A typical metal particle size suitable for use in materials such as CPG is about 50-150 nm.

CPG is widely used as a support matrix for chemical synthesis. Several stages of different particle size (CPG is usually treated as a fine particle powder), pore size (typically about 100 nm to about 1 μm), and surface derivatization (to promote chemical adhesion to the surface Various functional groups) can be used. CPG has a wide internal surface area (up to 100 m ² / g or more) and is chemically inert (unless derivatized to improve its metal binding or particle seeding properties), further causing spectral interference. It is particularly suitable as a biosensor support matrix because it does not cause the resulting substantial Raman peak.

  The pores should be large enough for the dye to enter and to accommodate the metal parts / particles. Typical dyes have a diameter of about 1-5 nm and analytes have a diameter of 5-100 nm. If metal particles with a diameter of about 50 nm are used, this means that the pore size should be about 150 nm or more.

  As an example, one method for making a silver-based substrate is to perform a Tollens reaction in the presence of CPG. Surface derivatization of CPG with aldehyde, alcohol, or carboxylate groups is particularly suitable for this method.

  In all embodiments of the invention, the support material is porous to the analyte to be detected and the detection chemical reaction (such as displacement of the reporter molecule or dye by the analyte) can occur within the support structure itself. However, this is not a requirement of the present invention. The support material is porous only to the dye depending on the analyte to be detected, and may not be porous to the analyte or other materials in the sample. Other materials may include anything in the sample that is not desirable for detection or that is desired to be separated from the irradiated part. Examples may include blood cells or chemicals that can distort the Raman signal. The analyte can displace the dye on the outer surface of the support material, which can then diffuse into the support material and interact with the Raman enhancing metal particles. The minimum requirement is that the support material be porous to the dye. Furthermore, the substrate particles are porous to the dye, but not to other substances in the analyte sample. The analyte can be on the external surface of the support material, or within the support material, but the pigment outside the substrate particles can be displaced, and then the dye diffuses into the substrate particles and Raman enhanced metal particles Interact with.

  If the support structure or substrate particle is porous only to the dye, the analyte may displace the dye from the selective agent on the outer surface of the support material or substrate particle. The dye will then diffuse into the support material or substrate particles and interact with the silver particles dispersed therein. Of course, if the analyte is also the dye itself, there is no need for a separate dye, and the support material or substrate particles will be porous to the analyte itself. When introduced into the reaction support, the analyte will diffuse into the support material or substrate particles and interact with the silver particles.

  The pore / pore size of the support material or substrate particles controls the distribution of the Raman enhancing particles throughout the material and the distance traveled for the dye to move and interact with the Raman enhancing surface. As a result, the pore / pore diameter of the entire support structure should be on the same order of magnitude as the mean free path of the dye used. The required diameter may be determined from the diffusion coefficient shown in FIG.

  The distribution of Raman enhancing particles within the pores should preferably be such that the distance between neighboring metal particles is of the same order of magnitude as the mean free path of the dye at which the Raman reaction is detected.

  When a substance such as CPG having a large internal surface area is used, the distance that interacts with the metal particles can be large because the molecules diffuse and the pores in the particles can be from 100 nm up to 10,000 nm. is there. When depositing metal in such a structure using methods such as crystallization or deposition, the distribution of metal particles takes a radial distribution function. Further, as it moves into the structure, fewer metal particles interact with the dye. However, the dye also diffuses in a similar manner, so that the majority of the dye molecules will be found within the areas with high concentration of metal particles.

  A three-dimensional porous support according to the present invention experiences two levels of resonance when illuminated by a laser. The first is surface plasmon resonance experienced by individual metal particles. In addition, it is a large-scale resonance that arises essentially from the metal placed in the dielectric medium (sample and support material). However, this second level of resonance shares similarities with resonances in the photonic crystal structure, but the diffusion across the metal particles in the support structure is not uniform and is therefore difficult to compare directly.

  A support structure having a metal dispersed and fixed therein, such as silver-doped CPG, may be considered equivalent to a physically fixed colloidal dispersion in a reasonable estimate (for calculation purposes only). . The sample solution in the CPG and pores approximates a dielectric medium with metal nanoparticles that line the inside of the CPG internal pore walls that are equivalent to colloidal particles in suspension. The pore size and the spatial dispersion of the nanoparticles on the pore wall determine the concentration of the colloid analog.

  The optional properties of colloidal particles have been extensively studied. The absorption spectrum of the colloid can be calculated from Mie theory. For small spherical particles, the absorbance A of the dispersion of N particles per unit volume depends only on the dipole term of the Mie sum and can be calculated as follows:

ここで、Ｃおよびｌは、それぞれ吸収横断面と光路長である。粒子寸法が伝導電子の平均自由行程よりも小さい場合、これらの電子は、粒子壁と「衝突」し、バルク物質よりも低い有効平均自由行程を提供する。

Here, C and l are an absorption cross section and an optical path length, respectively. If the particle size is smaller than the mean free path of conduction electrons, these electrons “collide” with the particle wall, providing a lower effective mean free path than the bulk material.

  Under the restriction of 2πR <λ (where R is the particle size and λ is the wavelength of light in the surrounding medium), the cross-sectional area can be expressed as:

ここで、Ｖは、球状粒子の体積、λは、入射波長（振動数ωに対応）、およびε_ｍは、媒体の誘電率である。バルク金属の複素相対誘電率は、以下のように求められる。

Here, V is the volume of the spherical particles, λ is the incident wavelength (corresponding to the frequency ω), and ε _m is the dielectric constant of the medium. The complex relative dielectric constant of the bulk metal is obtained as follows.

銀等の自由電子金属に対し、ε_１（ω）およびε_２（ω）は、よく知られている場合が多く、ある範囲の波長にわたって実験的に決定されている。上述の式より、最大値は、ε_１（ω）＝−２ε_ｍの場合の吸光度において生じることが分かる。ε_１（ω）＝−２ε_ｍの場合の値ε_２（ω）および波長に伴う変化率ε_１（ω）は、結果として生じる吸収バンドの高さおよび幅を決定する要因である。

For free electron metals such as silver, ε ₁ (ω) and ε ₂ (ω) are often well known and have been experimentally determined over a range of wavelengths. From the above equation, it can be seen that the maximum value occurs in the absorbance when ε ₁ (ω) = − 2ε _m . The value ε ₂ (ω) for ε ₁ (ω) = − 2ε _m and the rate of change ε ₁ (ω) with wavelength are factors that determine the height and width of the resulting absorption band.

  For metal particles having a diameter corresponding to the mean free path of conduction electrons (L), the collision rate between the free electrons and the particle wall is considerable. Electron motion is effectively damped, leading to changes in the dielectric properties of the metal. In order to consider this surface effect, the second term needs to be added to the calculation of the imaginary part of the dielectric function.

ここで、ε_２’（ω）は、小粒子に対し補正された誘電パラメータである。

Here, ε _{2 ′} (ω) is a corrected dielectric parameter for small particles.

  As mentioned above, typical silver particles are tens of nanometers in diameter if they can be dispersed on and within the support material provided by the present invention. A generally acceptable value for the mean free path L of electrons in silver is about 57 nm. The wavelength dependent values of the composite dielectric function for silver, glass, and water (approximate to the sample) are well known in the art. Using these data, the UV / visible absorption spectrum of silver-added CPG can be predicted.

  Comparison between theory and experimental results is shown in FIG. 25, but the experimental absorbance at short wavelengths is lower than the predicted value. This difference can be easily corrected by subtracting a Gaussian function of the form

以下に示される実験データに対する最適適合は、ａ＝７ｘ１０^−６、ｂ＝３．３４ｘ１０^−７、およびｃ＝３．５ｘ１０^−８によって求められる。

The best fit for the experimental data shown below is determined by a = ⁷ × 10 ⁻⁶ , b = 3.34 × 10 ⁻⁷ , and c = 3.5 × 10 ⁻⁸ .

  The parameter a is simply a magnification. Parameters b and c determine the position and width of the Gaussian peak, respectively. Subtracting this Gaussian function has the effect of providing a “notch” filter in the optical spectrum. This effect can be rationalized if the silver-doped CPG behaves like a photonic crystal with the band gap eliminated at about 334 nm. Experimental data was collected using a laser with a nominal wavelength of 532 nm (in vacuum). The nominal versus apparent wavelength ratio (532/334) provides an effective refractive index of 1.593 for the silver-doped CPG “photocrystal”. The refractive indices of glass, water, and silver at this nominal wavelength are about 1.520, 1.336, and 0.002, respectively. Accordingly, the silver-added CPG acts as a metamaterial having characteristics different from the component substances.

  The geometrical dispersion of silver particles within the CPG is not a regular crystal lattice, so conventional theories describing photonic crystals are not necessarily true. However, there is some local regularity in particle dispersion, which is mainly determined by the pore size and the pore gap spacing within the CPG structure. In this situation, the particles are not filled by a homogeneous dielectric medium, but by a sample (a dilute aqueous solution with uncertain dielectric properties containing at least the dye to be detected, and possibly the analyte, and other chemicals not detected). This is further complicated by the fact that they are spaced apart by a combination of open pores and, in this case, pore gap material comprising glass. The exact dielectric properties of such materials are highly dependent on the exact shape and spatial configuration of the CPG pores, and it is difficult to derive a general analytical explanation. However, the behavior of the silver-added CPG matrix can be predicted using numerical techniques such as finite element analysis or mesh-free methods.

  Thus, the optical behavior of the sensing substance is determined by two basic mechanisms. The first surface plasmon resonance is mainly influenced by the diameter, composition, and shape of the metal particles themselves. The second large-scale resonance is affected by the composition and spatial distribution of the support matrix, which determines the spatial distribution and dielectric environment in which the metal particles are dispersed. The optical properties of the metal particles themselves can be predicted using the mathematical techniques described above (and thereby rationally designed). The optical properties of the metal-added matrix material can be determined by analytical or numerical approaches, depending on the complexity of the structure. The different refractive indices and dielectric properties of the support material and the sample determine the optimum spacing between the metal particles that is required to ensure the maximum absorbance of the incident radiation. The average spacing of the metal particles is ideally half the wavelength of the incident radiation, however the wavelength depends on the material through which the radiation passes. The pore diameter ratio with respect to the wall thickness of the support material is preferably equal to the ratio of the refractive index of the solution filling the pores to the refractive index of the support material, in which case the actual wavelength of the excitation photons in both materials can be the same. . As a result, the pore / wall structure dimensions in the CPG metamaterial should preferably be selected as a weighted average taking into account the difference between the volume of support material with pores and the volume with solid support material. . A spacing of metal particles that is a multiple of half the wavelength of the incident radiation will also provide additional resonance harmonics.

  Lasers used in Raman spectroscopy typically have a focal region of about 100 μm, but this may be expanded to a size of 500 μm. FIG. 18 shows that the CPG particle size may be about 100 microns in total length. Thus, it should be understood that the support material according to the present invention can comprise a single substrate particle having a porous structure with the metal fixed and dispersed therein. Ideally, as few particles as possible are used because many particles effectively dilute the sample concentration. For convenience, more particles may be used to simplify detector design / configuration incorporating the present invention.

  Typically, lasers are used in Raman spectroscopy, but the only requirement is that the illumination source be a single color with a specified wavelength. The irradiation source need not be coherent.

  The pore diameter can affect the light irradiation if the pore diameter is approximately the same as the wavelength of light used. By using a pore size that is sufficiently smaller than the wavelength of light, these challenges are reduced.

  The particle size and shape have a dramatic effect on the ability of the metal particles to couple with the incident laser via resonance of the conduction electrons at the surface with the plasma (plasmons). Experimental evidence for this is shown in FIG. 22 (extracted from Haes et al., May 2005 MRS Bulletin (30)).

  FIG. 22 shows the UV / visible absorbance profile of a surface coated with particles of a given diameter and shape. The most complete data set currently available, covering both the UV / visible and infrared regions, is for triangular particles and is shown in the table below.

非線形回帰モデルを三角形粒子データに適用することによって、λｍａｘと粒子幅および高さとの関係を示す実験式が以下のように導出され得る。

By applying a non-linear regression model to triangular particle data, an empirical equation showing the relationship between λmax and particle width and height can be derived as follows.

これは、測定値に対しプロットされた計算値λｍａｘを示す図２３に示されるように、実験データに対し合理的に良好な適合を提供する。

This provides a reasonably good fit to the experimental data, as shown in FIG. 23 which shows the calculated value λmax plotted against the measured value.

  This relationship applies strictly to triangular particles and is based on a relatively small number of experimental data points, so if more observations are available, its accuracy is greatly improved. It will be. In particular, its accuracy will be very low for combinations of width and height that deviate significantly from the experimentally observed range. However, considering these points, it is possible to predict estimated λmax values for silver particles of various widths and heights. The dark curve in the plot of FIG. 24 shows better binding to the commonly used 532 nm laser.

  FIG. 24 is not symmetrical with respect to width and height because the observed particle system relates to a two-dimensional planar array particle. The particle field interacts significantly in the plane of the surface, not in the out-of-plane direction, so “width” relates to the in-plane dimensions of the particle and “height” relates to its diameter perpendicular to the plane.

  Undesirably, there are particle widths (approximately 100 nm) with λmax greater than 532 nm. Particles of relatively narrow width (<about 50 nm) have an undesirably low predicted value for λmax. For particle widths greater than about 100 nm, the optimum height of resonance with a 532 nm laser is (height≈25 + 0.42 × width). Particles with a width of about 75 nm have an optimum height below 50 nm and particles with a width below 75 nm have a sub-optimal λmax value below 532 nm. Therefore, the size within which a particle of a particular size attached to the surface will exhibit good light attenuation for a specified laser wavelength is in a narrow range.

  Particles formed by deposition using the Tollens reaction have a range of different diameters that are dispersed by fractal dispersion. One advantage of such a range of diameters is to ensure that there is a certain percentage of particles with the appropriate diameter to provide an enhancement in the response of the dye to radiation from radiation. . In Raman spectroscopy, this works with a plasmon wavelength that matches the wavelength of the incident radiation. However, if only particles of the required diameter are evenly dispersed on the surface of the substrate, the surface enhanced Raman effect will be further increased. This can be achieved by fixing the silver colloid in the support material. This is because such colloid diameter is much more controllable than silver particles produced through deposition techniques.

  Assuming that the particles deposited by the Tollens reaction approximate particles with a = b = observed diameter, the surface enhanced Raman signal from the population with the size distribution shown in FIG. It can be inferred that it is brought about by 0.6%. This number is in good agreement with the experimentally observed incidence of so-called “hot particles” which are considered to be less than 1%. However, it should be noted that a uniform population having a diameter approximating the above-mentioned optimum value has a very high percentage of hot particles, resulting in a significant improvement in the Raman signal for a given amount of silver.

(Explanation of rationale)
FIG. 1 shows a known microfluidic SER® S sensor. The illumination laser is focused onto a planar silver surface in a chamber on the microfluidic chip, thereby sampling the defined surface patch. Molecules absorbed on the surface in this region scatter photons by the Raman mechanism, and a certain percentage of the scattered light returns to the spectrometer recording the spectrum. For surface enhanced resonance Raman spectroscopy (SERRS), an incident laser that matches the absorbance peak of the Raman scattering molecule is selected, thereby improving the efficiency of generating a Raman signal. The use of a laser with a wavelength of 532 nm is typical because it is inexpensive and readily available.

  One of the important effects conferred by microfluidic systems is a dramatic reduction in reaction time by physically limiting the reaction to a volume in a dimension comparable to the diffusion path length of the relevant molecule.

  The ability of a molecule to diffuse through a liquid medium is explained by its diffusion coefficient D and can be predicted using the Stokes-Einstein equation as follows:

ここで、ｋは、Ｂｏｌｔｚｍａｎｎ定数、Ｔは、絶対温度である。このモデルは、分子が連続溶媒内を自由に拡散する半径Ｒの球体であって、分子径は、溶媒径の少なくとも５倍であって、液体は、低粘度（η）を有すると仮定する。

Here, k is a Boltzmann constant, and T is an absolute temperature. This model assumes a sphere of radius R in which molecules freely diffuse in a continuous solvent, the molecular diameter is at least 5 times the solvent diameter, and the liquid has a low viscosity (η).

  Proteins are not perfectly spherical, but their apparent Stokes radius can be determined experimentally. Exemplary examples are shown in the table below.

これらのデータは、その分子量から蛋白質のストークス半径を予測するための経験的方法を導出するために使用可能である。

These data can be used to derive an empirical method for predicting the Stokes radius of a protein from its molecular weight.

  FIG. 2 shows a plot of Stokes radius plotted against molecular weight. The experimentally determined point is on a logarithmic curve according to the relationship R = 1.27119ln (molecular weight) −1.6908. Using this equation, it is possible to use the predicted value of R in the Stokes-Einstein equation to predict the diffusion coefficient of any protein based only on its molecular weight. A plot showing the calculated diffusion coefficient plotted against molecular weight can be seen in FIG.

A general method for predicting a typical diffusion limited time scale (t _D ) in a microfluidic system uses the following equation:

ここで、ｌは、系の特性長である。ｌ＝１ｃｍを有するマクロスケール系内の質量１００ｋＤａの典型的蛋白質に対し、ｔ_Ｄは、約２３０時間である。ｌ＝１００μｍを有するマイクロ流体系内の同一蛋白質に対し、ｔ_Ｄは、約８４秒である。

Here, l is the characteristic length of the system. For a typical protein of mass 100 kDa in a macroscale system with l = 1 cm, t _D is about 230 hours. For the same protein in a microfluidic system with l = 100 μm, t _D is about 84 seconds.

  “Typical time scale” calculations show that microfluidic biosensors can have a significant effect on assay time, provided that reaction detection has dimensions comparable to the diffusion pathway of molecular components. It is carried out in a reaction chamber having. However, this calculation does not take into account the decrease in molecular components with binding events at the surface of the molecular sensor. A more realistic model of a biosensor can be determined using Fick's first law of diffusion for particle flux j due to a concentration gradient.

図４は、バイオセンサモデルの略図を示し、検体分子はＳｔｏｋｅｓ−Ｅｉｎｓｔｅｉｎモデルによって流体内に拡散し、それらは高さｈの反応チャンバ内に密閉され、その選択剤（抗体または核酸オリゴマー等の結合パートナーとしても知られる）は１つの壁に付着されると仮定される。また、右から放出するのと同じくらい多くの分子が左から画定領域に侵入するため（厳密には、チャンバ側壁から大幅に離れた領域に対してのみ当てはまる仮定）、側方拡散は重要ではないと仮定される。計算を単純にするために、選択剤への結合は不可逆的であって、過剰な結合パートナーが存在し、それぞれ個々の結合事象は瞬間的であると仮定される。

FIG. 4 shows a schematic diagram of a biosensor model, where analyte molecules diffuse into the fluid by the Stokes-Einstein model, which is sealed in a reaction chamber at a height h, and its selective agent (such as an antibody or nucleic acid oligomer binding Is also assumed to be attached to one wall. Also, lateral diffusion is not important because as many molecules enter the defined region from the left as to release from the right (strictly speaking, this applies only to regions that are far away from the chamber sidewall) Is assumed. To simplify the calculations, it is assumed that binding to the selective agent is irreversible, there are excess binding partners, and each individual binding event is instantaneous.

The analyte concentration at height x and time t is defined as c (x, t). The initial state (t = 0) has c (x, t) = C ₀ (initial concentration), and the decrease in the coupling wall is c (h, t) = 0 (where x = h). . As it progresses, the binding reaction reduces the concentration of the analyte in proximity to the binding wall. This includes a diffusion flux of analyte molecules towards this wall, as a concentration gradient is formed and, over time, the chamber is depleted of analyte molecules.

  The calculation is performed in two stages, shown schematically in FIG. In stage 1, there is a decreasing region close to the wall where x = h and is continuously replenished by diffusion of molecules from the rest of the chamber. In stage 2, this reduced area grows to include the entire chamber. One additional estimate used in the calculation is to assume that the concentration gradient has a linear profile.

  The first step is to calculate the time required for stage 1. From Fick's law, the diffusion to the coupling wall is derived by the diffusion gradient over the decreasing region, which is assumed to have a linear profile.

チャンバ内の時刻ｔにおける自由検体分子の数ｎは、以下によって求められる。

The number n of free analyte molecules in the chamber at time t is determined by:

次いで、変化率（すなわち、フラックス）は、以下となる。

Next, the rate of change (ie, flux) is as follows.

境界条件ｘ（０）＝０を使用すると、以下となる。

If the boundary condition x (0) = 0 is used:

時刻ｔ_１で生じる段階１の最後に、ｘ（ｔ_１）＝ｈとなり、したがって、以下となる。

At the end of phase 1 occurring at time t ₁ , x (t ₁ ) = h, and thus:

段階２の計算は同様である。再び、結合壁に対する拡散を考慮するが、今回は、減少領域を考慮する必要があるだけである。

The calculation of stage 2 is similar. Again, we consider diffusion to the coupling wall, but this time we only need to consider the reduced region.

次いで、自由検体分子の数が、以下によって与えられる。

The number of free analyte molecules is then given by:

したがって、フラックスは、以下となる。

Therefore, the flux is as follows.

境界条件ｃ（０，０）＝Ｃ_０を使用すると、以下が求められる。

Using the boundary condition c (0,0) = C ₀ , the following is obtained:

これは、指数関数的減衰であるため、ゼロに達するまで検体濃度に対し無限時間をとることになる。しかしながら、検体の一定の割合ｐの結合に要する時間を計算可能である（０＜ｐ＜１）。これをｔ_２と呼ぶ。

Since this is an exponential decay, it will take an infinite time for the analyte concentration until it reaches zero. However, it is possible to calculate the time required for binding a certain proportion p of the analyte (0 <p <1). This is referred to as _{t 2.}

したがって、以下となる。

Therefore:

段階２に指数関数的減衰式を使用すると、以下であることが分かる。

Using an exponential decay equation for stage 2, we can see that:

次いで、検体分子の割合ｐの結合に要する時間ｔ_ｐは、以下のように与えられる。

Then, the time t _p taken for the binding of the percentage of analyte molecules p is given as follows.

すなわち、

That is,

この式をマイクロ流体系に対し容認される「典型的時間スケール」式と比較すると、以下となる。

Comparing this equation with the “typical time scale” equation accepted for microfluidic systems:

チャンバ高ｈが、特性長ｌとなり、検体減少のため系に導入される指数関数的減衰を説明する追加項が存在することは明白である。

Obviously, there is an additional term that accounts for the exponential decay introduced into the system due to the chamber height h becoming the characteristic length l and analyte reduction.

  For a typical 100 kDa protein, the time required to achieve 99% maximum binding of microfluidic reaction chambers of different heights can be calculated, and a plot of chamber height versus time at which 99% binding occurs is shown in FIG. Shown in

  The plot of FIG. 6 uses a log-log scale because the numerical range of both axes spans several orders of magnitude. For chamber heights below about 40 μm, binding is complete in seconds. For heights between 40-310 μm, 99% bonding is achieved in minutes, and for heights above 310 μm, bonding takes several hours. A typical microtiter plate has a length scale of a few millimeters corresponding to a bonding time of over 10 hours, which means that the plate is not subject to vibration or agitation during bonding that can greatly accelerate the process. Assuming

  The calculated time course of binding over an hour for an example of a typical 100 kDa protein is shown in FIG. 7 at different chamber heights. The effect of reducing the length scale is obvious. For a “macroscale” of 1 cm in height, less than 5% binding is achieved after 1 hour, but at a chamber height of 100 μm, binding is essentially complete after about 6 minutes.

FIG. 1 shows a conventional microfluidic SER® S sensor. FIG. 2 shows a plot of the Stokes radius plotted against the molecular weight of various proteins. FIG. 3 shows the calculated diffusion coefficients for various proteins plotted against molecular weight. FIG. 4 shows a schematic diagram of a known biosensor model. FIG. 5 shows plots used to calculate analyte concentrations at various depths in the biosensor model of FIG. FIG. 6 shows a plot of chamber height against the time at which 99% binding of the analyte to the binding partner occurs. FIG. 7 shows the calculated amount of binding of a typical 100 kDa protein over 1 hour for different chamber heights. FIG. 8 shows a microfluidic SER (R) S sensor embodying the present invention. FIG. 9 shows the focusing characteristics of a laser focused on the mirror (left) and in the volume (right) that can be used in the embodiment of the invention of FIG. FIG. 10 shows a particulate three-dimensional support matrix in the form of a filter frit. FIG. 11 shows filter frit silica particles coated with particulate metallic silver that may be employed in embodiments of the invention such as FIG. FIG. 12 schematically shows the silica particles of the filter frit of FIG. 11 coated with particulate metallic silver. FIG. 13 shows a deposited silver morphology comprising filamentary strands formed by a first deposition method according to aspects of the present invention. FIG. 14 shows a second form of deposited silver. FIG. 15 shows the increased silver deposition per unit area obtained by the second deposition method according to a further aspect of the present invention. FIG. 16 shows superimposed silver particle size plots obtained by the two deposition methods together. FIG. 17 shows an exemplary SERRS spectrum from particles prepared on a silica filter, as shown in FIG. 11, using two different methods. FIG. 18 shows pore glass particles with a diameter of about 80-120 μm. FIG. 19 shows an enlarged view of the surface of a single porous glass particle. FIG. 20 is an enlarged view of the surface of a single porous glass particle at a higher magnification than FIG. 19 and shows a pore size of about 200-500 nm. FIG. 21 shows metal particles that can be deposited in the pores shown in FIG. FIG. 22 shows the UV / visible absorbance profile of a surface coated with particles of a given diameter and shape. FIG. 23 shows the calculated value λmax plotted against the measured value. FIG. 24 shows the predicted λmax values for silver particles of various widths and heights. FIG. 25 shows the absorbance of the support material according to an embodiment of the invention compared to the theoretical value for a given emission wavelength. FIG. 26 shows the plot of FIG. 25, where an additional term was used in the theoretical calculation.

Claims (47)

A reaction carrier into which a test sample can be introduced for use in a detector configuration that detects the presence, absence, or amount of an analyte in the sample,
A solid support material configured to define a volume;
A metal dispersed within the volume and supported by the support material,
A reaction support, wherein the support material is porous to the dye and the metal is configured to enhance the light response to irradiation of the dye.   The reaction carrier according to claim 1, wherein the metal is configured to enhance the response to irradiation by interaction between electrons in the metal and the dye.   The reaction carrier according to claim 1 or 2, wherein the detected response is a SERS interaction.   The reaction carrier according to claim 1 or 2, wherein the detected response is a SERRS interaction.   The support material is configured to have the dye dispersed within the volume, and the response to irradiation is such that the dye is dispersed within the volume and is similarly dispersed within the volume. 5. The reaction carrier according to any one of claims 1 to 4, which is enhanced as a very close result.   The reaction carrier according to any one of claims 1 to 5, wherein the metal is dispersed in the volume as a plurality of dispersed solid metal portions.   The reaction carrier according to claim 6, wherein the dispersed metal portion is a metal particle.   The reaction carrier according to claim 6 or 7, wherein the support material is composed of one or more base particles, and the metal is deposited on an external surface of the one or more base particles.   The reaction support according to claim 6, 7, or 8, wherein the metal is deposited within the one or more substrate particles.   The dye is attached to a selective agent capable of binding the analyte within the volume, but when the analyte is bound to the selective agent, the dye moves to the region near the metal. The reaction carrier according to any one of claims 1 to 9.   The detector arrangement of claim 10, wherein the dye is displaceably attached to the selective agent and held away from the metal by the selective agent.   The reaction carrier according to claim 9, wherein the base particle is porous to the dye.   10. A reaction carrier according to claim 9, wherein the substrate particles are porous to the dye but not to other substances in the analyte sample.   The reaction carrier according to claim 7, wherein the distance between neighboring particles of the metal is about the mean free path of the dye.   15. The reaction carrier according to any one of claims 1 to 14, wherein the support material does not produce a response to irradiation within a frequency range of the response to irradiation of the dye.   The reaction carrier according to claim 1, wherein the specimen to be detected functions as the dye.   The reaction carrier according to any one of claims 1 to 16, wherein the support material is composed of silica.   The reaction carrier according to any one of claims 1 to 17, wherein the support material is composed of CPG.   19. A reaction carrier according to any one of claims 1 to 18, wherein the response to irradiation is an intensity change at a given wavelength shift due to Raman scattering.   20. The reaction carrier according to any one of claims 1 to 19, wherein the metal type is selected to enhance the response to irradiation of the dye.   The reaction carrier according to any one of claims 1 to 20, wherein the metal is silver, gold, or copper.   The reaction carrier according to claim 7, wherein the metal particles have a dimension of the approximately mean free path of electrons in the metal. A reaction carrier according to any one of claims 1 to 22, and
An irradiation source;
A detector;
A pigment and
The irradiation source is configured to irradiate the reaction carrier with radiation of a specified wavelength and to detect the absence, presence, or amount of a dye in response to irradiation indicative of the absence, presence, or amount of an analyte Container configuration.   24. The detector arrangement of claim 23, wherein the illumination source is configured to provide illumination that covers at least a portion of a volume defined by a support material.   25. The detector arrangement of claim 24, wherein the illumination source is focused so that the portion of the volume includes the rays of the illumination source.   26. The detector arrangement of claim 25, wherein the portion of the volume includes light rays of the illumination source on either side of the focal point of the illumination source.   The detector configuration of claim 7, wherein the dimension of the metal particles is about 10-250 nm.   8. A detector arrangement according to claim 7, wherein the size of the metal particles and the wavelength of irradiation radiation are such that a significant proportion of incident radiation is absorbed by the metal particles.   8. A detector arrangement according to claim 7, wherein the spatial dispersion of the metal particles and the wavelength of the irradiation radiation are such that a significant proportion of incident radiation is absorbed by the metal particles.   30. The detector configuration of claim 29, wherein the spatial dispersion of the metal particles is approximately equal to half the wavelength of the irradiating radiation within the volume.   30. The detector configuration of claim 29, wherein the spatial dispersion of the metal particles is approximately equal to any multiple of half the wavelength of the irradiating radiation within the volume. A method for detecting the presence, absence or amount of a sample analyte in a reaction carrier comprising:
A solid support material configured to define a volume within the reaction support;
Providing a metal dispersed and fixed in the volume and supported by the support material, wherein the support material is porous to the dye and the metal reacts to irradiation of the dye A step configured to enhance, and
Providing a dye; and
Irradiating the reaction support with radiation of a specified wavelength;
Detecting a response to irradiation from the dye and determining an amount of the dye indicative of the presence, absence or amount of the analyte.   The dye is attached to a selective agent capable of binding the analyte, but when the analyte sample is introduced, the analyte binds to the selective agent, and the selective agent moves the dye to the region near the metal. 35. The method of claim 32, wherein   34. The method of claim 33, wherein the selective agent holds the dye away from the metal until the introduction of an analyte that peels the dye and moves to the near metal region.   35. In response to introduction of the analyte sample, the analyte can bind to the selective agent that strips the dye and diffuses into the support material, thereby migrating to the near metal region. The method in any one of.   The support material is composed of one or more base particles, the metal is deposited in the one or more base particles, and the specimen peels the dye in response to the introduction of the specimen sample. 36. The method of claim 35, wherein the method can bind to the selective agent that diffuses into the one or more particles and thereby migrate to the near metal region.   The support material is porous to the dye, but not to other materials, so that when the analyte displaces the dye, only the dye can diffuse into the support material. The method according to any one of 35 to 36.   The support material is porous to the dye, but the one or more substrate particles are not porous to the analyte, whereby only the dye is displaced when the analyte displaces the dye. 38. The method of claim 36, wherein can diffuse into the one or more substrate particles.   39. A method according to any one of claims 32 to 38, comprising introducing the analyte sample into the reaction carrier that allows the dye to enter the volume.   39. A method according to any one of claims 32 to 38, comprising introducing the analyte sample into the volume. 41. A method according to any one of claims 32 to 40, comprising irradiating at least a portion of the volume defined by the support material with the irradiation source.   42. The method of claim 41, wherein the illumination source is a laser and comprises configuring the laser such that the portion of the volume includes a beam of the laser.   43. The method of claim 42, wherein the portion of the volume includes the laser beam on either side of the laser focus.   The dye is attached to a selective agent capable of binding the analyte, but is retained in the region near the metal, and when the analyte sample is introduced, the analyte binds to the selective agent, and the selective agent 35. The method of claim 32, wherein the dye can be separated from the near metal region.   45. The method of claim 44, wherein the selective agent retains the dye in the near metal region until the introduction of an analyte that peels the dye and separates from the near metal region.   Within the volume, the dye is attached to a selective agent capable of binding the analyte and is held in the region near the metal. When the analyte is bound to the selective agent, the dye is the metal. The reaction carrier according to claim 1, which is separated from a nearby region.   47. The detector configuration of claim 46, wherein the dye is movably attached to the selective agent and is retained in the near metal region by the selective agent.

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